Microvascular segment extraction method and application thereof

Abstract

The invention relates to the field of biomedicine, and discloses a capillary segment extraction method and application thereof. The method comprises a step of digesting rat brain tissues and a step of separating brain microvascular segments. The method provided by the invention is simple to operate, can be directly realized on wild animals, and does not depend on animal endogenous fluorescent labeling. According to the method, the complete vascular tissue can be obtained, meanwhile, the activity of the cells is reserved, the method is the first method capable of separating the complete vascular tree from the brain tissue and then further researching the functions of all the cells on the vascular tissue, and the method has important significance on research of the brain microvascular segment.

Description

Technical Field

[0001] This application relates to the biomedical field, and more specifically, to a method for extracting microvascular segments and its uses. Background Technology

[0002] Neurovascular units are crucial structures ensuring nutrition and communication between blood vessels and brain tissue. They play a role in regulating the blood-brain barrier and are involved in the pathophysiological processes of diseases such as acute cerebral infarction, chronic cerebral ischemia due to arterial stenosis, and cerebral small vessel disease. Neurovascular units are composed of cerebral microvascular endothelial cells, pericytes, microglia, neurons, and extracellular matrix. Exploring the pathophysiological changes of these cells and intercellular signaling is an important method for improving the prognosis of patients with acute brain injury.

[0003] In view of this, many studies have reported the use of mechanical grinding-gradient centrifugation, fluorescence flow cytometry sorting, and microdissection to separate brain vascular cells from cortical tissue and enrich brain vascular cells for subsequent analysis; however, the existing technology still has the following technical problems: (1) Mechanical separation is prone to causing damage to vascular cells, affecting subsequent experiments that require the collection of live cells; mesh filtration causes a large loss of blood vessels, both of which easily reduce the cell separation yield; (2) Fluorescence flow cytometry sorting requires gene-edited mice carrying specific fluorescent marker genes, which is cumbersome and limits its use in other species without fluorescent markers. Furthermore, the method of enriching by using marker genes is prone to selection bias; (3) Microscopic dissection requires extremely high technical skills from the experimenter, and the diameter of the obtained blood vessels is limited, and microvessels with a diameter of less than 40um (such as capillaries, arterioles and venules) cannot be obtained; (4) In the current reported studies on brain microvascular gene expression profiles, most studies focus on a sorted cell population (such as endothelial cells), and there are few methods that can obtain complete microvascular segments and completely analyze all vascular structural cells and vascular attachment cells, which limits the study of signal communication changes in different cell types of brain microvessels.

[0004] In summary, isolating intact microvascular segments from adult rodent brain tissue while preserving cellular viability has always been a challenge in neurobiology. Developing a method for isolating intact microvascular segments from adult rat brain tissue while preserving all cell types and attached immune cells would not only effectively overcome the shortcomings of existing technologies but also be of great significance for subsequent experiments such as single-cell sequencing and mapping the gene expression atlas of all cell types in brain microvessels. Summary of the Invention

[0005] The purpose of this invention is to develop a gentler method for isolating microvascular segments in the brain of adult rats. This method is gentler than existing mechanical grinding methods, preserves the immune cells attached to the microvascular segments, obtains brain microvessels of all diameter levels, has no selection bias on all brain microvascular structures and attached cells, and ensures the viable cell rate in the obtained brain microvascular segments, thus enabling it to be adapted for subsequent research that requires live brain microvascular cells.

[0006] To achieve the above-mentioned objectives, this application adopts the following technical solution: In a first aspect, this application provides a method for extracting microvascular segments, comprising the following steps: Step 1, digesting rat brain tissue: 1-1. Dissection and separation of rat cortex: Fresh brain tissue was taken, the pia mater and related major arteries were removed, the cerebral cortex was dissected and cut into small pieces; 1-2. Place the shredded brain tissue in DMEM medium containing 1% collagenase and digest it by shaking at 37°C. Repeat the pipetting operation during digestion. After pipetting is completed, continue digestion until no visible tissue fragments are visible. Step 2, separating brain microvascular segments: 2-1. Add DMEM medium to stop digestion, centrifuge and discard the supernatant; 2-2. Add phosphate buffer containing 25% bovine serum albumin to the precipitate, gently pipette and invert to mix, centrifuge, collect the bottom layer of microvascular segment precipitate, wash, and obtain brain microvascular segments.

[0007] Furthermore, in steps 1-2, the multiple blowing and beating operations include: First pipetting: Use a 1ml pipette tip with a cut end; Second blow-drip: using a standard Pasteur straw; Third blow-through: Use a standard 1ml suction tip.

[0008] Furthermore, each blowing operation is spaced 20 minutes apart.

[0009] Furthermore, after the third blowing is completed, digestion continues for 15 minutes.

[0010] Further, in steps 1-2, the shredded brain tissue is placed in 10 times its volume of DMEM medium containing 1% collagenase for digestion and dissociation.

[0011] Furthermore, in step 2-1, the centrifugation conditions are: 4°C, 1000g, centrifugation for 5 minutes.

[0012] Furthermore, in step 2-2, the centrifugation conditions are: 4°C, 2000g, centrifugation for 15min.

[0013] Further, in step 2-2, the washing process is as follows: add PBS and gently mix by pipetting, centrifuge at 800g for 5 minutes at 4°C, discard the supernatant, and repeat the washing until pure vascular segment tissue is obtained.

[0014] Secondly, this application provides the use of the described microvascular segment extraction method in transcriptomics, proteomics, metabolomics, epigenomics, and single-cell analysis.

[0015] In summary, this application has the following beneficial effects: The separation method established in this application first dissociates the complete structure of microvascular segments from brain tissue, and then further dissociates the complete microvascular segments into single cells and connects them to the subsequent sequencing process. This method can unbiasedly preserve the microvascular segments and all cell types attached to them, avoiding the cell selectivity caused by certain markers in the previous flow cytometry sorting method, and helps to discover new and special microvascular-related cell types.

[0016] The process of dissociating microvascular segments in this application is gentle and, compared to previous brain vessel dissociation methods based on grinding, better preserves the cell viability of microvascular segments, meeting the cell viability requirements for single-cell sequencing. Furthermore, this dissociation method effectively preserves immune cells attached to the vascular segments, facilitating further research into specific subsets of microvascular-associated immune cells.

[0017] This application incorporates a step-by-step digestion and agitation process into the enzymatic dissociation of brain tissue, and tests the effect of prolonged digestion time on tissue viability. Verification shows that this method overcomes the limitation of previous enzymatic dissociation methods, which were too mild and could only be applied to the brain tissue of young mice, successfully adapting it to the study of adult and aged mouse brain tissue, and providing a new extraction method for the study of microvessels in the adult rat brain. Attached Figure Description

[0018] Figure 1 : Experimental flowchart of an embodiment of this application.

[0019] Figure 2 Light microscopy and immunofluorescence images. As shown in the images, a complete vascular tree morphology is visible under the light microscope. Figure 2 a); Lectin, as a vascular marker, can be used to complete the labeling of vascular trees ( Figure 2 (b) As a marker of vascular smooth muscle, a-SMA shows that the lower main vascular trunk consists of arterioles with smooth muscle structures, while the upper, smaller, dendritic vessels are the downstream capillary tree. Figure 2 c). Detailed Implementation

[0020] The technical solutions and effects of this application will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0021] Example 1: Extraction of microvascular segments This embodiment discloses a method for extracting microvascular segments, including the following steps: Step 1, digesting rat brain tissue: ① Dissection and separation of rat cortex: Take freshly separated brain tissue from rats over 4 months old, roll it on absorbent paper to remove the pia mater and related major arteries, dissect the cerebral cortex, and then use Venus scissors to cut the cerebral cortex into small pieces of 1 cubic millimeter.

[0022] ② Digestion and Dissociation: Rat brain tissue was placed in ten times its volume of DMEM medium containing 1% collagenase and gently shaken for digestion at 37°C. During digestion, the mixture was gently pipetted every 20 minutes using a 1ml pipette: for the first pipetting, a large opening was cut off from the 1ml pipette; for the second pipetting, a standard Pasteur pipette was used; for the third pipetting, a standard 1ml pipette tip was used, and the mixture should be able to be pipetted and aspirated without resistance after this third pipetting. After the three pipetting cycles were completed, digestion continued for 15 minutes. No visible tissue fragments should be visible after digestion.

[0023] Step 2, separating brain microvascular segments: ① Pour the mixture into a 15ml centrifuge tube, add DMEM culture medium to bring the volume to 15ml, and stop the digestion.

[0024] ② Centrifuge at 1000g for 5 minutes at 4°C, discard the supernatant. The lower layer, containing charged tissue, is obtained. ③ Add 10 ml of phosphate-buffered saline (PBS) containing 25% (w / w) bovine serum albumin (BSA) to the tissue, gently pipette and invert to mix, strictly avoiding the formation of air bubbles. Then centrifuge at 4°C and 2000g for 15 minutes.

[0025] ④ The bottom red precipitate after centrifugation is the microvascular segment. After discarding the upper nerve tissue and the middle BSA solution, transfer the bottom vascular segment precipitate to a new centrifuge tube, add PBS, and gently mix by pipetting.

[0026] ⑤ Washing: Centrifuge at 4°C, 800g for 5 minutes, and discard the supernatant. This step can be repeated until relatively pure vascular segment tissue is obtained for subsequent analysis.

[0027] Example 2: Verification of Vascular Segment Tissue (1) Immunofluorescence histology: ① Fixation: Add the vascular segment tissue to 5ml of 4% paraformaldehyde, mix well, and let stand at room temperature for 20min for fixation.

[0028] ② Washing: Pour the paraformaldehyde containing the vascular segment tissue into a 40µm filter to filter out the paraformaldehyde, and then wash with at least 20ml of PBS. Invert the filter onto the opening of a 50ml centrifuge tube and backwash the filter with at least 10ml of PBS to collect the microvascular segment into the centrifuge tube. Centrifuge at 800g for 5 minutes at 4°C, and discard the supernatant.

[0029] ③ Membrane disruption: Add 3 ml of PBS containing 0.3% Triton X-100 and 5% (w / w) BSA, mix well, transfer to a 5 ml centrifuge tube, and let stand at room temperature for 30 min.

[0030] ④ Incubation with fluorescently labeled primary antibody: Centrifuge at 800g for 5 min at 25°C, and discard the supernatant. Add 1 ml of PBS containing 1 μl of FITC-labeled α-SMA antibody (Sigma, F3777), 5 μl of DyLignt 594-labeled lectin (Vectorlab, DL-1177), 0.1% Triton X-100, and 5% (w / w) BSA. Mix well and incubate overnight at 4°C in the dark.

[0031] ⑤ Washing: Centrifuge at 800g for 5 minutes at room temperature, and discard the supernatant. This step can be repeated until the non-specific fluorescent label is washed away.

[0032] ⑥ Slide Preparation: Resuspend the cleaned vascular tissue in an appropriate amount of PBS, mix gently, and drop an appropriate amount of PBS containing the vascular tissue onto an adhesion slide. Let it stand until the vascular tissue adheres, then discard the excess PBS. After the slide has dried slightly, add an appropriate amount of anti-fluorescence quenching mounting medium and mount the slide.

[0033] (2) Immunofluorescence results: such as Figure 2 As shown in Figure a, the complete vascular tree morphology is visible under a light microscope. Lectin, as a vascular marker, can be used to label vascular trees (…). Figure 2 (b) As a marker of vascular smooth muscle, a-SMA shows that the lower main vascular trunk consists of arterioles with smooth muscle structures, while the upper, smaller, dendritic vessels are the downstream capillary tree. Figure 2 c). This method can better preserve the integrity of vascular tissue.

[0034] (2) Cell viability verification: This experiment was commissioned to Shanghai Liebing Biopharmaceutical Co., Ltd. The obtained vascular tissue was subsequently digested into single cells, and then subjected to various viability testing methods, including trypan blue, achieving a viability rate of over 80%. Subsequent single-cell sequencing analysis revealed the separation of various cell groups, including endothelial cells, pericytes, vascular smooth muscle cells, and fibroblasts. The data quality was excellent, with an extremely low proportion of low-quality cells, suggesting that this method is suitable for various experiments requiring the preservation of vascular tissue cell viability.

[0035] The beneficial effects of this invention are: the method of this application introduces enzymatic dissociation, which combines collagenase digestion with gentle mechanical dissociation (three-stage blowing), while retaining the gentleness of enzymatic dissociation, and enhancing the dissociation force through gentle blowing. This solves the problem that enzymatic dissociation is too gentle and requires a very long digestion time in adult and old animal tissues, greatly shortening the dissociation time and reducing the stress of digestion on cells.

[0036] This method yields complete vascular tissue while preserving cell viability. It is the first method that allows for the separation of a complete vascular tree from brain tissue before further studying the function of individual cells within the vascular tissue. This is of great significance for the study of brain microvascular segments.

[0037] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for extracting microvascular segments, characterized in that, Includes the following steps: Step 1, digesting rat brain tissue: 1-1. Dissection and separation of rat cortex: Fresh brain tissue was taken, the pia mater and related major arteries were removed, the cerebral cortex was dissected and cut into small pieces; 1-2. Place the shredded brain tissue in DMEM medium containing 1% collagenase and digest it by shaking at 37°C. Repeat the pipetting operation during digestion. After pipetting is completed, continue digestion until no visible tissue fragments are visible. Step 2, separating brain microvascular segments: 2-1. Add DMEM medium to stop digestion, centrifuge and discard the supernatant; 2-2. Add phosphate buffer containing 25% bovine serum albumin to the precipitate, gently pipette and invert to mix, centrifuge, collect the bottom layer of microvascular segment precipitate, wash, and obtain brain microvascular segments.

2. The method for extracting microvascular segments according to claim 1, characterized in that, In steps 1-2, the multiple blowing and beating operations include: First pipetting: Use a 1ml pipette tip with a cut end; Second blow-drip: using a standard Pasteur straw; Third blow-through: Use a standard 1ml suction tip.

3. The method for extracting microvascular segments according to claim 2, characterized in that, Each blow-drying operation should be spaced 20 minutes apart.

4. The method for extracting microvascular segments according to claim 2, characterized in that, After the third blowing is completed, continue digestion for 15 minutes.

5. The method for extracting microvascular segments according to claim 1, characterized in that, In steps 1-2, the shredded brain tissue is placed in 10 times its volume of DMEM medium containing 1% collagenase for digestion and dissociation.

6. The method for extracting microvascular segments according to claim 1, characterized in that, In step 2-1, the centrifugation conditions are: 4°C, 1000g, centrifugation for 5 minutes.

7. The method for extracting microvascular segments according to claim 1, characterized in that, In step 2-2, the centrifugation conditions are: 4°C, 2000g, centrifugation for 15min.

8. The method for extracting microvascular segments according to claim 1, characterized in that, In step 2-2, the washing process is as follows: add PBS and gently mix by pipetting, centrifuge at 800g for 5 minutes at 4°C, discard the supernatant, and repeat the washing until pure vascular segment tissue is obtained.

9. Use of the microvessel segment extraction method according to any one of claims 1-8 in transcriptomics, proteomics, metabolomics, epigenomics and single-cell analysis.

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